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Azadirachtin Biosynthesis Induction in Azadirachta indica A. Juss


Cotyledonary Calli with Elicitor Agents

Article  in  Brazilian Archives of Biology and Technology · April 2014


DOI: 10.1590/S1516-89132014000200001

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Vol.57, n.2: pp. 155-162, March-April 2014 BRAZILIAN ARCHIVES OF


ISSN 1516-8913 Printed in Brazil
BIOLOGY AND TECHNOLOGY
A N I N T E R N A T I O N A L J O U R N A L

Azadirachtin Biosynthesis Induction in Azadirachta indica A.


Juss Cotyledonary Calli with Elicitor Agents
Marcelo Rodrigues1*, Reginaldo Alves Festucci-Buselli2, Luzimar Campos Silva3 and
Wagner Campos Otoni3
1
Departamento de Biologia; Setor de Fisiologia Vegetal; Universidade Federal de Lavras; Lavras - MG - Brasil.
2
Centro de Tecnologia Agropecuária; Instituto Sócio Ambiental e dos Recursos Hídricos; Universidade Federal
Rural da Amazônia; 66077-530; Belém - PA - Brasil. 3Departamento de Biologia Vegetal; Universidade Federal de
Viçosa; Viçosa - MG - Brasil

ABSTRACT
The use of cell and plant tissues culture techniques to produce economically important active metabolites has been
growing. Among these substances, azadirachtin (AZA), produced by the neem tree (Azadirachta indica), has
received considerable attention due to its bioinsecticide action. The main goal of this work was to analyze the AZA
levels in neem cotyledonary calli. The calli were grown in agitated Woody Plant Medium (WPM) liquid medium,
supplemented with glucose (Gl), hydrolyzed casein (HC) and methyl jasmonate (MeJ) as elicitor agent. An
interaction was observed between these substances, depending on in vitro cultivation time with orbital agitation.
The highest concentrations (average of 0.2470 µg g-1) of AZA were produced in the first and second weeks of
culture when the cell mass was grown in a medium with 2% Gl v/v, 500 mg L-1 HC and 100 µM of MeJ. This
corresponded to approximately 57% of the AZA content stored in the donor plants seeds, used as a source of
explants to induce in vitro callus formation. It was concluded that the nutrition, as well as the concentration of MeJ
as signal transduction of secondary metabolism in neem cells, might influence the AZA content produced in vitro.

Key words: Neem, HPLC, glucose, hydrolysed casein, methyl jasmonate

INTRODUCTION reforestation projects, or as an ornamental tree for


landscaping.
The neem tree (Azadirachta indica A. Juss), syn The neem tree produces more than 300 secondary
Antela azadirachta, or Melia azadirachta L. metabolites, a third of which are tetratriterpenoids
belongs to the Meliaceae family (Cunha 2002). It (limonoids) with different biological effects and
is originated from Asia, native to Burma and arid commercial applications (Dai et al. 2001; Neves et
regions of the Indian subcontinent, where there are al. 2003; Rodrigues et al. 2012). The best known
approximately 18 million trees. It is currently active compounds are azadirachtin (AZA), an
grown in Australia, Africa and the Americas also. insecticide and insect repellent, nimbin, a
According to the US National Council of spermicide and anti-inflammatory in vertebrates,
Investigation, neem has become one of the most and salanim, another insect repellent. Of these,
promising plants worldwide for its various AZA (Fig. 1), which is stored mainly in the leaves
applications (Kundu 1999). It is used in human and seeds Thengane et al. (1995), is the most
and veterinary medicine, in the cosmetics industry, economically important and abundant metabolite
as green manure, as a hardwood, and also for produced by A. indica (Mordue and Blackwell
*
Author for correspondence: marcel.or.7@hotmail.com

Braz. Arch. Biol. Technol. v.57 n.2: pp. 155-162, Mar/Apr 2014
156 Rodrigues, M. et al.

1993). AZA may cause changes in the insect metabolite biosynthesis. The discovery of new
development by repellent and intoxicating effects, compounds and their elicitors may allow the
affecting the physiology, oviposition and egg manipulation of their biosynthetic routes,
viability (Chopra 1958; Neves et al. 2003). increasing their yield in the tissue and cell cultures
Observations of the feeding behavior of some (Kim et al. 2009). To increase the yield, many
larvae (Spodoptera littoralis and Mamestra scientists have used a technique based on the
brassicae) indicated that AZA might also reduce application of elicitors to a cultivation medium.
the food consumption (Neves et al. 2003). This causes stress in the plant cells, signaling and
activating defense mechanisms in the plants.
Commonly used elicitors include carbohydrates,
jasmonic acid (JA), methyl jasmonate (MeJ),
salicylic acid (SA), chitosan and heavy metal ions
(Nascimento and Fett Netto 2010).
The aim of this study was to assess in vitro
production of AZA, using neem cotyledonary calli
in agitated liquid medium cultures under the
influence of different elicitor agents: glucose (Gl),
hydrolysed casein (HC) and methyl jasmonate
(MeJ).

Figure 1 - The AZA molecule structure (C35H44O16) is a


tetratriterpenoid produced by Azadirachta MATERIALS AND METHODS
indica, mainly stored in seeds and leaves.
Callus induction
Neem plants (Azadirachta indica A. Juss)
AZA is a complex molecule that would be difficult germinated in vitro were used as explant source.
to synthesize chemically. Thus, the cell and tissue Seeds were collected at the Nim Brazil nursery
culture approaches are the best options for in vitro garden in São José do Rio Preto, SP, Brazil.
production of these metabolites. The use of Cotyledons were inoculated on semisolid Woody
biotechnology may optimize the production of Plant Medium - WPM Lloyd and McCown (1981)
bioactive compounds through the use of elicitor with 6 g L-1 granulated agar (Merck®, Germany),
agents and other methods (Prakash et al. 2002; and 3 mg L-1 6-benzyladenine (BA) and 0.5 mg L-1
Festucci-Buselli et al. 2008; Nakabayashi et al. indolyl-3-butyric acid (IBA) (adapted
2010). However, AZA has not always been easy to methodology from Rodrigues et al. 2009), pH to
detect using the standard techniques. For instance, 5.8. The medium was sterilized by autoclaving at
Akula et al. (2003) was unable to detect the AZA 120ºC during 15 min Aliquots (10 mL) were added
in somatic embryos of neem using HPLC, even to the test tubes (25 X 150 mm) and closed with
though the extract of somatic embryos was known transparent polypropylene lids.
to affect the development of locust larvae. This
showed that somatic embryos synthesized the Callus cultivation
AZA, or another metabolite that affected the insect The calli were cultured in the flasks (90 mm
development. These apparent problems in HPLC height, 50 mm diameter sealed with rigid
analysis by UV detection may be caused by the poplyproplylene lids), containing 40 mL of
low sensitivity of the detector, which lacks a semisolid WPM medium, added with 3 mg L-1 BA
strong-UV absorbing molecular chromophore. and 0.5 mg L-1 IBA, and sub-cultured every 20
Such low sensitivity could cause errors in the days. Six calli were transferred to each flask, with
quantification of AZA (Schaaf et al. 2000). the same medium, growth regulators and
Secondary metabolites are produced by the plants concentrations, added with 200 mg L-1 myo-
mainly under stress conditions. The stresses may inositol, 0.5 g L-1 polyvinyl pyrrolidone (PVP), 2%
trigger the plant defense mechanisms, leading to a sucrose v/v, and B5 vitamins Gamborg et al.
variety of biochemical signals, which induce the (1968) until the obtention of fresh mass calli for
expression of genes related to secondary setting the experiment. The medium pH was
adjusted to 5.8 previously to autoclaving at 120°C

Braz. Arch. Biol. Technol. v.57 n.2: pp. 155-162, Mar/Apr 2014
In vitro Production of Azadirachtin 157

during 15 min. The calli were cultivated in the the injected volume for each run was 50 µL. The
dark. Three samples of cotyledonary calli were run time was 30 min. The mobile phase was
kept in 125 mL Erlenmeyer flasks with 40 mL of filtered with 0.45 µm Millipore membrane and
WPM liquid medium, with the same growth degassed with helium. The data were integrated by
regulators and concentrations and placed on an the Shimadzu SPD software. To set the calibration
orbital shaker at 90 rpm. curve, AZA with 95 % purity (Sigma Aldrich,
EUA) was used at concentrations of 0; 0.0125;
Cultivation with inducing agents 0.025; 0.05; 0.1; 0.2 and 0.4 µg L-1 in methanol.
The agitated WPM liquid cultures were added with The resulting chromatogram values were plotted
the following combinations of elicitors and and a linear equation (Fig. 2) was used to calculate
evaluated for in vitro AZA production: 1- glucose the AZA content of the samples. The natural
(Gl) concentrations as carbon source (0, 1, 2, extract of neem seeds was analyzed under the
and 4 % v/v); 2 - hydrolyzed casein (HC) identical conditions.
concentrations as nitrogen source (0, 250, 500,
1000 mg L-1); 3 - methyl jasmonate (MeJ) Experimental design
concentrations as elicitor (0, 50, 100, 200 µM) A simple factorial design (3x4) was used,
versus time. Three treatments were used: T0 comparing the concentrations of AZA and the
(control); T1 (1% Gl v/v + 250 mg L-1 HC + 50 elicitor agents over different collection weeks,
µM MeJ); T2 (2% Gl v/v + 500 mg L-1 HC + 100 with nine replicates per treatment. The data were
µM MeJ); and T3 (4% Gl v/v + 1000 mg L-1 HC + submitted to the variance analysis of the GENES
200 µM MeJ). Weekly periodic sampling (W1, software, version Windows/2004.2.1 to Cruz
W2, W3 and W4) was conducted. At the end of (2002), using the Tukey post-hoc test with two
each week, three Erlenmeyers were sampled from factors and a 5% significance level.
each of the four treatments (12 Erlenmeyers in
total). The dry mass was calculated after storage in
an oven at 60oC for 24 h, establishing a default RESULTS AND DISCUSSION
value of 0.75 g per Erlenmeyer collected. The
neem seeds were milled and dried to obtain the dry Azadirachtin is a tetraterpenoid and is the most
mass, mixed with methanol, filtered and injected abundant molecule in neem seeds when compared
into High Performance Liquid Chromatography to other limonoids. The detected AZA content of
(HPLC) in order to compare the AZA content the seed extract was higher than the cultivated calli
produced in the treatments with natural levels. when analyzed with a UV wavelength of 217 nm.
Such contrasts could occur when there was a
Methanol soluble extract obtainment differential in vitro gene expression of several
Callus dry mass and seed dry mass were divided neem secondary metabolites. Differences in the
into three 0.25 g portions and stored in an gene expression are caused by the somaclonal
Eppendorf with 1.0 mL of methanol per sample. variation in the explants during the sub-cultures
After 24 h, the samples were centrifuged at 3000 (Kota et al. 2006; Lima et al. 2008). On the other
rpm for 10 min, and subsequently injected into hand, this somaclonal variation can be a useful
HPLC to determine the level of AZA present in source of new variation for genetic breeding,
the methanol soluble extract. mainly for the selection of cell lines with higher
yield (Carvalho et al. 2013). The a appearance of
AZA content determination the calli (Fig. 2A) cultivated in the semisolid
AZA content analysis was performed using the medium prior to liquid medium as well as the
HPLC (Shimadzu SPD-M10AVP Diode Array AZA calibration curve is shown in Figure 2B. A
Detector) equipment, following the method two-phase system was used for the induction of
described by Schaaf et al. (2000), with some the rapid cell growth. Growth regulators were used
modifications. Ultraviolet (UV) was used as and then the cells were exposed to a liquid
detector with a 217 nm wavelength. A Bondesil medium containing elicitor agents: Gl as carbon
C18 reverse phase column (5 µm x 4.6 mm x 250 source, HC as nitrogen source and MeJ as signal
mm) was used, with 1.0 mL min-1 flow and transduction of secondary metabolism in neem
column pressure at 97 Kgf. The mobile phase cells in vitro.
solvent was methanol and water (50:50 v/v), and

Braz. Arch. Biol. Technol. v.57 n.2: pp. 155-162, Mar/Apr 2014
158 Rodrigues, M. et al.

Figure 2 - Azadirachta indica cotyledonary calli subcultured every 30 days in Woody Plant Medium -
WPM. A) Calli from the ninth subculture in semisolid medium, then transferred to liquid
medium; B) Linear regression of the AZA calibration, with R2 = 0.999.

The AZA production process can be optimized, when


two phases are taken. The first phase is characterized by
high nitrogen and phosphorus contents to increase the
biomass. The second phase uses low concentrations of
minerals, inducing the nutritional stress in the explants
and reducing the oxygen transference rate (OTR).
Consequently, there may be increase in the AZA
production. According Raval et al. (2003), the highest
AZA concentration (0.8 mg L-1 extract) was related
with the cells at the ninth day of cultivation in White’s
Medium (White 1963). However, after applying this
two-phase system, the highest AZA concentration Figure 3 - HPLC chromatogram. Retention time of the
produced in vitro reached 67.5% (2.7 mg/L-1 extract) in standard – 95% purity AZA (Sigma Aldrich,
callus cultures from the shoots, using the modified USA): b = 13.617 min, dotted line; treatment 2
Murashige and Skoog (MS) medium with different and week 2 (T2W2 = 2% Gl v/v + 500 mg L-1
concentrations of nitrogen and phosphorus sources in HC + 100 µM MeJ): a = 13.525 min in solid
suspension culture callus, relative to the average line.
content of the compound in natural seeds (Raval et al.
2003). These results indicated that the biomass growth
was not directly related to the in vitro production of
AZA, but directly related to the decrease of the OTR
associated with the stationary phase of cell growth. The
nutrient type and its concentration are primordial to the
cell expansion and development.
Based on the chromatogram (Fig. 3), the standard AZA
retention time was 13.617 min, with higherer
production of AZA in second week, when the cell was
cultivated in WPM liquid medium with 2% Gl v/v +
500 mg L-1 HC and 100 µM of MeJ (T2W2) took
13.525 min. By superposition, the analyzed compound
was considered AZA due to the similarity of the
retention time of the standard and sample. Figure 4 - AZA concentration (µg per gram of callus dry
There was significant interaction between the elicitor mass) as affected by elicitor agents: T1 [1% glucose (Gl)
agent concentrations as a function of sample collection v/v + 250 mg L-1 hydrolized casein (HC) + 50 µm methyl
time. The highest AZA concentration was produced by jasmonate (MeJ)]; T2 [2% (Gl) v/v + 500 mg L-1 (HC) +
the T2 treatment with 2% Gl v/v, 500 mg L-1 HC and 100 µM (MeJ)] and T3 [4% (Gl) v/v + 1000 mg L-1 (HC) +
100 µM MeJ (Fig. 4). 200 µM (MeJ)], as a function of the weekly collection (W1
to W4).
The lowercase letters indicate differences among weeks in
a same concentration of elicitors agents and the uppercase
letters indicate differences for a week among
concentrations, at 5% by the Tukey's test.

Braz. Arch. Biol. Technol. v.57 n.2: pp. 155-162, Mar/Apr 2014
In vitro Production of Azadirachtin 159

The first and second weeks had the highest important factor in the production of secondary
production rates with an average AZA metabolites by the plant cell cultures
concentration of 0.247 µg per gram of callus dry (Chattopadhyay et al. 2002).
mass in the fifth subculture at 15 days intervals. Nitrate nitrogen is a better nitrogen source than
The control samples (without elicitor agents) ammonia for biomass production and for
resulted AZA only in traces, and therefore, no area stimulating AZA accumulation in vitro. The
integration was performed (in chromatograms). maximum biomass was 15.02 g L-1 calli and 2.98
Apparently increased production of bioactive mg/g-1 of AZA per gram of callus, 12 days after
compounds with use of elicitor agents, may relate the cultivation of the suspension culture. Seed
to the “Rate Growth Hypothesis” Stamp (2003), kernel (along with embryo) was, thereafter,
which suggests that when the maximum growth excised and used as an explant for callus induction
decreases, the rates from the constitutive defense on MS medium, optimizing the nutritional
levels increase. The lower amount of carbon and composition of the culture medium and additives.
nutrients assimilated by the cell – related to the Indeed, the high ammonium concentration
increase in osmotic potential – cause an increase in inhibited the cell growth and AZA production
secondary metabolites production. The AZA (Prakash and Srivastava 2005). According to
content of the treatment T2W2 (2% Gl v/v + 500 Sujanya et al. (2008), 94.41 mm of nitrate, 23.60
mg/L-1 HC + 100 µM MeJ) corresponded to 57% mm (4:1) of ammonium and a reduction in sucrose
of the AZA stored in the seeds derived from the content (15 mg L-1) increased the AZA production
adult neem plants (0.4301 µg g-1). (5.59 mg L-1) in MS medium with 1.0 mg L-1 NAA
Abiotic stresses can affect the chemical and 3.0 mg L-1 BA. However, a biomass reduction
composition and amount of essential oils produced (7.4%) was also recorded when the cell
by the plants. The biochemical and chemical suspensions were established from the nodal
agents may favor the biochemical signaling of the segments. Some solutes of low molecular weight
different metabolic pathways the production of such as ammonium, aminoacids (proline) and
bioactive compounds (Guillon et al. 2006). The poliols (sorbitol and mannitol), sugars (sucrose,
perception of the elicitor signal leads to the glucose, fructose and threalose), are considered
activation of transduction factors that regulate the osmoprotectors because they are highly soluble
expression of the genes involved in the and directly influence the synthesis and
biosynthesis of secondary metabolites. These accumulation of secondary metabolites in plants
substances can also modify the conformation, or (Berenguer et al. 2008; Suriyan et al. 2009).
activation of kinase receptors by activating their Nutrition is known to directly influence the
effectors, such as ion channels, protein G, and content of AZA, mainly through its impact on
lipases, which are responsible for translating the carbon sources (Prakash et al. 2002). MeJ and JA,
elicitor signal into the plant’s defense response. in their role as signal transduction elements in the
The locations of the ligation of elicitor signals pathways of secondary metabolites, have been
have been identified in the plasma membrane of used to induce the production of economically
various plant species. These signals can be important compounds. MeJ has some limitations
biochemical or chemical, and are able to for long term induction of secondary metabolites
effectively influence the synthesis of secondary in certain crops. For example, the cells of Silybum
metabolites in vitro (Zhao et al. 2005). marianum elicited with MeJ for the production of
Glucose is a better carbon source than sucrose for silimarin showed a gradual decrease in the level of
biomass production and is capable of stimulating this compound throughout the successive
higher production of the AZA, because glucose is subcultures (Sanchez-Sampedro et al. 2009).
a monosaccharide that has better access to the Ohyama and Nitsch’s (1972) culture medium
cells, being a primary energy source to the cells. produced the highest in vitro yield of AZA
Apparently, sucrose, after being taken up from the (0.0166% dry mass). Adding elicitor agents such
culture media, was hydrolyzed to glucose and as JA (100 mm) and SA at 100 mm, resulted in 6-
fructose by the cell wall bound invertases folds (0.095%) and 9-folds (0.14%) increase,
(Martinez and Park 1993). The slow consumption respectively in the production of AZA in neem
of sucrose might be due to low activity of the root explants (Satdive et al. 2007). Biotic and
invertase. The selection of carbon source (glucose, abiotic elicitor agents have been recommended as
or sucrose) has long been recognized as an an important strategy to optimize the in vitro

Braz. Arch. Biol. Technol. v.57 n.2: pp. 155-162, Mar/Apr 2014
160 Rodrigues, M. et al.

generation of secondary metabolites (Savitha et al. an orbital agitated WPM liquid medium
2006). supplemented with 3.0 mg L-1 BA, 0.5 mg L-1
JA is a signaling agent for the production of IBA, 2% Gl v/v + 500 mg L-1 HC, and 100 µM
bioactive compounds in the plants, which undergo MeJ.
mechanical stress by the insects (Prakash and
Srivastava 2008). Thus, AZA production and
content observed in this work were probably ACKNOWLEDGEMENTS
influenced by the nutrition system in the culture
medium and the concentration of agents used to The authors thank Dr. Everaldo Gonçalves de Barros
stimulate its biosynthesis. Elicitors agents such as for making available laboratory facilities for HPLC
chitosan, SA, JA, MeJ and yeast extract have been analyses, and to Eduardo Rezende Pereira for his
used in neem cell suspension to promote higher technical assistance. MR was recipient of a scholarship
levels of the AZA in vitro production. The from CNPq.
combination of these agents could enable up to 5-
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